CN115531921A - Preparative liquid chromatography fraction collecting device and method - Google Patents
Preparative liquid chromatography fraction collecting device and method Download PDFInfo
- Publication number
- CN115531921A CN115531921A CN202211032628.0A CN202211032628A CN115531921A CN 115531921 A CN115531921 A CN 115531921A CN 202211032628 A CN202211032628 A CN 202211032628A CN 115531921 A CN115531921 A CN 115531921A
- Authority
- CN
- China
- Prior art keywords
- liquid chromatography
- collection
- sample
- preparative liquid
- preparative
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 53
- 238000004262 preparative liquid chromatography Methods 0.000 title claims abstract description 23
- 238000001228 spectrum Methods 0.000 claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 230000014759 maintenance of location Effects 0.000 claims abstract description 12
- 239000000523 sample Substances 0.000 claims description 64
- 238000001802 infusion Methods 0.000 claims description 26
- 238000002347 injection Methods 0.000 claims description 20
- 239000007924 injection Substances 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 16
- 239000002699 waste material Substances 0.000 claims description 13
- 238000000862 absorption spectrum Methods 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 6
- 238000002835 absorbance Methods 0.000 claims description 5
- 238000004237 preparative chromatography Methods 0.000 claims 2
- 239000000126 substance Substances 0.000 abstract description 20
- 230000008569 process Effects 0.000 abstract description 12
- 238000001514 detection method Methods 0.000 abstract description 8
- 238000012545 processing Methods 0.000 abstract description 6
- 206010063385 Intellectualisation Diseases 0.000 abstract description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000010828 elution Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 230000003595 spectral effect Effects 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/24—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the treatment of the fractions to be distributed
- B01D15/247—Fraction collectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/12—Circuits of general importance; Signal processing
- G01N2201/129—Using chemometrical methods
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Health & Medical Sciences (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a preparative liquid chromatography fraction collecting device and a preparative liquid chromatography fraction collecting method.A detection part adopts a spectrum signal of a diode array detector as a collecting judgment basis and collects the fraction according to the triple judgment conditions of spectrum similarity, peak purity and peak height; interference substances with the same retention time can be eliminated through qualitative judgment of the spectrum similarity; the collection component can be ensured to be a single substance through the quantitative judgment of the peak purity value; the concentration of the collected components can be ensured to meet the requirements through the quantitative judgment of the peak height. The invention has the advantages of no need of human intervention, high automation, high collection accuracy and good repeatability. The intellectualization, the automation and the operability of the system are improved, and the process of secondary discrimination processing after collection is omitted, so that the prepared liquid chromatogram fraction is more efficiently and accurately collected.
Description
Technical Field
The invention relates to the field of high performance liquid chromatography, in particular to a preparative liquid chromatography fraction collecting device and a preparative liquid chromatography fraction collecting method.
Background
At present, with the development of analytical instruments, liquid chromatography is widely applied in the fields of life sciences, traditional Chinese medicines, biochemical engineering, medicines, scientific research and the like as a method for effectively realizing sample recovery or sample purification and preparation.
The fraction collector is an auxiliary device of a semi-preparative/preparative liquid chromatograph, and is mainly used for collecting components after chromatographic separation. The fraction collector can collect the chromatographic peak signals in a manual or automatic control mode. The automatic control mode has the characteristics of intellectualization and high collection efficiency, and gradually replaces the traditional manual collection mode. But the accuracy of distinguishing direct influence components of chromatographic peak signals in the automatic collection process and the further identification or processing result of subsequent fractions. Therefore, a good collected signal discrimination plays a crucial role in the whole fraction collection process.
The following methods are commonly adopted for the chromatographic signal discrimination at present:
1) And (4) a manual mode. Experimenters observe the peak state of the chromatogram through eyes and judge the falling point of the chromatographic peak. When the collection condition is met, the collection port is manually moved to a collection bottle for collection, and the waste liquid port is moved back after the collection is finished. Due to the adoption of a manual intervention mode, a large error exists in the collection process, and the collection effect cannot be ensured.
2) Collected over time. In the initial stage of the experiment, the conditions are firstly groped to find the common peak time of chromatographic peaks. The collection method, i.e. the collection start and end times, are set according to the obtained empirical values during the actual experiment. Because the starting time and the ending time are fixed values, the time of the chromatographic peak changing along with the conditions of the environment temperature, the state of the liquid chromatographic system and the like. Therefore, the collecting and distinguishing mode according to time cannot ensure that the collected samples have the same accuracy, and the repeatability is poor among batches with large individual difference.
3) And collecting according to chromatographic threshold. This is a method for judging the collection condition according to the magnitude of the absorbance value of the sample. Firstly, setting a starting collection threshold value and an ending collection threshold value respectively according to the characteristics of a sample chromatographic peak. In the experimental process, when the absorption value of the chromatographic peak exceeds the initial threshold value, the collection is started, and when the absorption value is reduced to the end threshold value, the collection is stopped. This collection discrimination is true provided that the collected chromatographic peaks contain only a single component. The chromatographic peak collecting effect is poor for chromatographic peaks containing two or more similar components. After the collection is completed, identification and processing are performed through cumbersome steps.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a preparative liquid chromatography fraction collecting device and a preparative liquid chromatography fraction collecting method.A detection part adopts a spectrum signal of a diode array detector as a collecting judgment basis, and the collecting conditions are judged by the triple of spectrum similarity, peak purity and peak height; the method has the advantages of no need of human intervention, high automation, high collection accuracy, good repeatability and no need of secondary identification treatment.
In order to achieve the purpose, the invention provides the following technical scheme: a preparative liquid chromatography fraction collection method comprising the steps of:
s1: injecting a component to be collected control solution;
s2: collecting the ultraviolet visible absorption spectrum of the component to be collected;
s3: storing the component spectrum to be collected into a spectrum library;
s4: injecting a sample solution to be separated;
s5: starting to collect and return to zero for timing;
s6: collecting a chromatogram and a spectrogram of a sample to be separated in real time;
s7: when the retention time t n In accordance with a start-stop time range, i.e. t 1 ≤t n ≤t 2 Entering a collection and judgment stage;
s8: judging whether the solution meets the judgment condition or not, and entering the next step if the solution meets the judgment condition;
s9: the fraction collector divides the solution into collection bottles.
The invention is further configured to: the determination conditions described in step S8 include:
f1: comparing the real-time spectrogram with the spectrogram of the component to be collected, which is acquired in the step S2, to obtain a similarity value Sn, and comparing the similarity value Sn with a similarity threshold S set by the method 0 Comparing, and judging that Sn is more than or equal to S 0 ；
F2: calculating a peak purity value Pn according to the real-time acquired spectrogram and a peak purity threshold value P set by the method 0 Comparing, and judging that the condition Pn is more than or equal to P 0 ；
F3: obtaining a peak height value (absorbance value) Hn from a real-time acquired chromatogram, and a peak height threshold value H set by the method 0 Comparing, and judging that the Hn is more than or equal to H 0 。
The invention is further configured to: if any of the above determination conditions is not satisfied, the fraction collector remains connected to the waste liquid channel and does not perform collection.
The invention is further configured to: in the step S7, if the time t is reserved n If the start-stop time range is not met, the determination condition is not required, and the fraction collector is kept communicated with the waste liquid flow path and does not collect the fraction.
The invention is different from the prior automatic fraction collecting system adopting chromatographic signal to judge the peak, the detection part adopts the spectral signal of a diode array detector as the collecting and judging basis, and the invention has triple judging and collecting conditions of spectral similarity, peak purity and peak height. Compared with the chromatographic signal of the traditional single-wavelength detector, the method has the characteristic of qualitative detection. Wherein interfering substances with the same retention time can be excluded by qualitative judgment of the spectrum similarity. The collection of the fractions as a single species can be guaranteed by quantitative determination of the peak purity values. The concentration of the collected components can be ensured to meet the requirements through the quantitative judgment of the peak height. The intelligent, automatic and operable system is improved, the process of secondary judgment processing after collection is omitted, and real high efficiency and accuracy are achieved.
The invention is further configured to: a preparative liquid chromatography fraction collecting device comprises a first solvent bottle, a second solvent bottle, a sample injection pump, a mixer, a preparative chromatographic column, a diode array detector, a fraction collector and a data workstation.
The invention is further configured to: first solvent bottle, second solvent bottle respectively through infusion tube coupling mix the tee bend, mixed tee bend connect the blender, the infusion tube on still be provided with first transfer pump and second transfer pump respectively.
The invention is further configured to: the sample bottle is connected with the inlet of the sample injection pump through a liquid conveying pipeline, and the outlet of the sample injection pump is connected with a sample injection tee joint; the sample feeding tee joint is positioned at the downstream of the mixer; the outlet of the sample injection three-way is connected with the manual sample injection valve.
Through the technical scheme, the method can be realized as follows: mixing two mobile phases in the first solvent bottle and the second solvent bottle through a mixer, sucking a sample through a sample injection pump, mixing the sample with the mobile phases, and sending the mixture into a subsequent flow path system.
The invention is further configured to: the outlet of the manual sample injection valve is connected with the inlet of the preparative chromatographic column through a transfusion pipeline, and the outlet of the preparative chromatographic column is connected with the inlet of the diode array detector.
The invention is further configured to: the outlet of the diode array detector is connected with the fraction collecting inlet; and the outlets of all channels of the fraction collector are respectively connected with all collecting bottles.
The invention is further configured to: the data workstation respectively with sampling pump, first transfer pump, second transfer pump, diode array detector, fraction collector electric connection. The flow rates of the first infusion pump and the second infusion pump are respectively controlled through command signals; sampling volume of a sample pump; collecting a spectrum signal of a diode array detector; the fraction collecting valve is controlled to collect and discharge the fraction according to a predetermined method.
The technical scheme can realize that: separating the sample by a preparative chromatographic column, and separating different component substances after adsorption and elution in the preparative chromatographic column. Each component substance is detected in the diode array detector, the component meeting the requirement is opened by the fraction collector switching electromagnetic valve collecting channel, the designated component flows into each collecting bottle, and the collected component is not required to be discharged into a waste liquid channel.
In the working process, the data workstation is respectively connected with the sample injection pump, the first infusion pump, the second infusion pump, the diode array detector and the fraction collector through cables. The data workstation respectively controls the flow rates of the first infusion pump and the second infusion pump according to a set method. Through the control flow, full-automatic analysis and collection can be realized.
Firstly, S1: injecting a component to be collected control solution; the two mobile phases are stored in a first solvent bottle and a second solvent bottle, are respectively sucked by a first infusion pump and a second infusion pump, are converged at a mixing tee joint, and are fully mixed by a mixer. And then is fused with the component sample to be collected which is sucked by the sample pump in the sample introduction tee.
S2: collecting the ultraviolet-visible absorption spectrum of the component to be collected; separating the sample by a preparative chromatographic column, and separating different component substances after adsorption and elution in the preparative chromatographic column. The constituent species are detected in a diode array detector.
S3: storing the component spectrum to be collected into a spectrum library; the spectra generated in the diode array detector are stored in a spectral library in a data workstation.
S4: injecting a sample solution to be separated; the sample solution to be separated is prepared according to the method described in step S1.
S5: start acquisition and time to zero.
S6: collecting a chromatogram and a spectrogram of a sample to be separated in real time; separating the sample by a preparative chromatographic column, and separating different component substances after adsorption and elution in the preparative chromatographic column. And detecting each component substance in a diode array detector to generate a chromatogram and a spectrogram.
S7: when retention time t n In accordance with a start-stop time range, i.e. t 1 ≤t n ≤t 2 Entering a collection and judgment stage; if the retention time t n If the start-stop time range is not met, the determination condition is not required, and the fraction collector is kept communicated with the waste liquid flow path and does not collect the fraction.
S8: judging whether the solution meets the judgment condition; the data workstation distinguishes the generated chromatogram and the spectrogram according to the following conditions:
f1: comparing the real-time spectrogram with the spectrogram of the component to be collected, which is acquired in the step S2, to obtain a similarity value Sn, and comparing the similarity value Sn with a similarity threshold S set by the method 0 Comparing, and judging that Sn is more than or equal to S 0 ；
F2: calculating a peak purity value Pn according to the real-time acquired spectrogram and a peak purity threshold value P set by the method 0 Comparing, and judging that the condition Pn is more than or equal to P 0 ；
F3: obtaining a peak height value (absorbance value) Hn from a real-time acquired chromatogram, and a peak height threshold value H set by the method 0 Comparing, and judging that Hn is more than or equal to H 0 . If any of the above determination conditions is not satisfied, the fraction collector remains connected to the waste liquid channel and does not perform collection.
S81: if any of the above determination conditions is not satisfied, the fraction collector remains connected to the waste liquid flow path and does not perform collection.
S9: the fraction collector divides the solution into collection bottles. The data workstation sends the collection signal to the fraction collector, and the fraction collector switches solenoid valve collection channel and opens, and appointed component flows in formulating the receiving flask.
In addition to the above described automated sample collection, the preparative kit fraction collection system is compatible with conventional manual sample collection modes. During the use process, a user firstly switches the manual sampling valve to a sampling state, and a sample to be analyzed is input into the quantitative ring by adopting a large-volume injector. The volume of the manual sample introduction valve quantitative ring is usually 100mL, and quantitative rings of different specifications can be replaced according to the actual sample introduction amount of a user. And when the collection base line of the diode array detector is stable, switching the manual sample injection valve to a sample injection state. At the moment, the sample in the quantitative ring is brought into a system flow path under the action of the mobile phase, and the subsequent analysis and collection processes are the same as those of automatic sample introduction.
In summary, the technical scheme of the invention has the following beneficial effects:
1. the detection part adopts a spectrum signal of a diode array detector as a collection judgment basis and has triple judgment collection conditions of spectrum similarity, peak purity and peak height. Compared with the chromatographic signal of the traditional single-wavelength detector, the method has the characteristic of qualitative detection. Namely, the interference substances with the same retention time can be eliminated through the spectrum, and the collection result is more accurate. Meanwhile, the process of secondary discrimination processing after collection is omitted, and accuracy and high efficiency are really achieved.
2. The invention is also compatible with the traditional manual sample feeding mode. During the use process, the manual sample injection valve can be adjusted to collect the liquid chromatogram fraction.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of an application of an embodiment of the present invention;
FIG. 2 is a schematic diagram of a device connection according to an embodiment of the present invention;
FIG. 3 is a schematic circuit diagram of an embodiment of the present invention;
FIG. 4 is a diagram showing an ultraviolet-visible absorption spectrum of naphthalene as a component to be collected according to an embodiment of the present invention;
FIG. 5 is a peak purity curve for an embodiment of the present invention;
fig. 6 is an example of a collection method setup of a specific embodiment of the present invention.
In the drawings, the reference numbers indicate the following list of parts:
1-a sample bottle, 2-a first solvent bottle, 3-a second solvent bottle, 4-a sample pump, 5-a first infusion pump, 6-a second infusion pump, 7-a mixing tee, 8-a mixer, 9-a sample tee, 10-a manual sample valve, 11-a preparative chromatographic column, 12-a diode array detector, 13-a fraction collector, 14-a collection bottle and 15-a data workstation.
Detailed Description
In order to make the technical solutions of the present invention better understood, the following description of the technical solutions of the present invention with reference to the accompanying drawings of the present invention is made clearly and completely, and other similar embodiments obtained by a person of ordinary skill in the art without any creative effort based on the embodiments in the present application shall fall within the protection scope of the present application. In addition, directional terms such as "upper", "lower", "left" and "right" and the like in the following embodiments are directions with reference to the drawings, and thus, the directional terms are used for illustrating the present invention and are not intended to limit the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
The invention is further described with reference to the drawings and the preferred embodiments.
Example (b):
referring to fig. 1 and fig. 2, which are application flow charts of a preferred embodiment of the present invention, a preparative liquid chromatography fraction collection method is provided, which includes the following steps in combination with the apparatus structure diagram of the embodiment shown in fig. 2:
s1: injecting a component to be collected control solution; the two mobile phases are stored in a first solvent bottle 2 and a second solvent bottle 3, respectively sucked by a first infusion pump 5 and a second infusion pump 6, merged at a mixing tee 7, and fully mixed by a mixer 8. Then is fused with the component sample to be collected which is sucked by the sample feeding pump 4 in the sample feeding tee joint 9.
S2: collecting the ultraviolet-visible absorption spectrum of the component to be collected; the sample is separated through the preparative chromatographic column 11, and different component substances are separated after being adsorbed and eluted in the preparative chromatographic column 11. The constituent species are detected in a diode array detector 12.
S3: storing the component spectrum to be collected into a spectrum library; the spectra generated in the diode array detector 12 are stored in a spectral library in a data workstation 15.
S4: injecting a sample solution to be separated; the sample solution to be separated is prepared according to the method described in step S1.
S5: start acquisition and time to zero.
S6: collecting a chromatogram and a spectrogram of a sample to be separated in real time; separating the sample by the preparative chromatographic column 11, and separating different component substances after adsorption and elution in the preparative chromatographic column 11. The component substances are detected in a diode array detector 12 to generate a chromatogram and a spectrogram.
S7: when the retention time t n In accordance with a start-stop time range, i.e. t 1 ≤t n ≤t 2 Entering a collection and judgment stage; if the retention time t n If the start-stop time range is not met, the determination condition is not needed, and the fraction collector is kept communicated with the waste liquid flow path and does not collect the waste liquid.
S8: judging whether the solution meets the judgment condition; the data workstation 15 distinguishes the generated chromatogram from the spectrogram according to the following conditions:
f1: comparing the real-time spectrogram with the spectrogram of the component to be collected, which is acquired in the step S2, to obtain a similarity value Sn, and comparing the similarity value Sn with a similarity threshold S set by the method 0 Comparing, and judging that Sn is more than or equal to S 0 ；
F2: calculating the peak purity value Pn according to the real-time collected spectrogram and the peak purity threshold value P set by the method 0 Comparing, and judging that the condition Pn is more than or equal to P 0 ；
F3: obtaining a peak height value (absorbance value) Hn from a real-time acquired chromatogram, and a peak height threshold value H set by the method 0 Comparing, and judging that Hn is more than or equal to H 0 。
S81: if any one of the judging conditions is not satisfied, the fraction collector keeps communicating with the waste liquid flow path and does not collect the fraction;
s9: the fraction collector divides the solution into collection bottles. The data station 15 sends a collection signal to the fraction collector 13, and the fraction collector 13 switches the solenoid valve collection channel open and the specified component flows into the specified collection bottle 14.
Note that S 0 The similarity threshold set for the user in the chromatographic method parameters can be an empirical value or an index required by the user, and is a numerical value of a thousandth ratio, if the spectrums of the sample to be measured and the reference substance (target object) are very similar, and the similarity calculated by the two is higher than a numerical value, the two can be judged to be the same substance, and one of the collection conditions is met. Generally, according to experience, the similarity is higher than 950 permillage, and the similarity can be judged to be the same substances;
P 0 the peak purity threshold value set for the user in the chromatographic method parameters is also an empirical value or index, and is also a numerical value of one thousandth, if the substance giving the peak is a pure substance, the peak purity numerical value of the substance is higher than a certain value, for example, higher than 950 thousandth, so that the sample to be detected is judged to be the pure substance at the moment, namely one of the conditions for collecting components is met;
H 0 for the peak height threshold set in the chromatographic method parameter by the user, the higher the sample concentration is, the higher the peak height is, so the user needs to know in advance how much the peak value of the sample to be measured can reach, or the peak value is converted according to the concentration of the reference substance, so that the desired substance concentration is satisfied after how much the peak height reaches in the collection experiment.
t 1 And t 2 That is, the retention start-stop time required for collection calculation, for example, the peak of the component to be measured occurs at 10-12min, then t1 can be set to be 10min, and t2 can be set to be 12min, so that the spectrogram which peaks at other time periods does not need to perform the above-mentioned complicated determination calculation.
As shown in fig. 3, which is a schematic circuit connection diagram according to an embodiment of the present invention, the data workstation 15 is electrically connected to the sample injection pump 4, the first infusion pump 5, the second infusion pump 6, the diode array detector 12, and the fraction collector 13, respectively. The flow rates of the first infusion pump 5 and the second infusion pump 6 are respectively controlled through command signals; the sample injection volume of the sample injection pump 4; collecting the spectrum signal of the diode array detector 12; the fraction collector 13 is controlled to collect and discharge the fraction according to a predetermined method.
FIG. 4 shows a diagram of an ultraviolet-visible absorption spectrum obtained by using the method and apparatus of the present invention, taking naphthalene as an example of a component to be collected; FIG. 5 shows a peak purity curve for a specific embodiment of the present invention; FIG. 6 is an exemplary set of collection methods for embodiments of the present invention that use the component naphthalene.
In summary, the detection part of the present invention uses the spectrum signal of the diode array detector 12 as the collection judgment basis, and has triple judgment collection conditions of spectrum similarity, peak purity and peak height. Compared with the chromatographic signal of the traditional single-wavelength detector, the method has the characteristic of qualitative detection.
Wherein interfering substances with the same retention time can be excluded by qualitative judgment of the spectrum similarity. The collection of the fractions as a single material can be guaranteed by quantitative determination of the peak purity values. Quantitative judgment of peak height can ensure that the concentration of the collected components meets the requirement. The intelligent, automatic and operable system is improved, the process of secondary judgment processing after collection is omitted, and real high efficiency and accuracy are achieved.
All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to those skilled in the art without departing from the principles of the present invention should also be considered as within the scope of the present invention.
Claims (10)
1. A preparative liquid chromatography fraction collection method is characterized by comprising the following steps:
s1: injecting a component to be collected control solution;
s2: collecting the ultraviolet-visible absorption spectrum of the component to be collected;
s3: storing the component spectrum to be collected into a spectrum library;
s4: injecting a sample solution to be separated;
s5: starting to collect and return to zero for timing;
s6: collecting a chromatogram and a spectrogram of a sample to be separated in real time;
s7: when the retention time t n In accordance with a start-stop time range, i.e. t 1 ≤t n ≤t 2 Entering a collection and judgment stage;
s8: judging whether the solution meets the judgment condition or not, and entering the next step if the solution meets the judgment condition;
s9: the fraction collector divides the solution into collection bottles.
2. A preparative liquid chromatography fraction collection method according to claim 1, wherein the judgment condition in step S8 includes:
f1: comparing the real-time spectrogram with the spectrogram of the component to be collected, which is acquired in the step S2, to obtain a similarity value Sn, and comparing the similarity value Sn with a similarity threshold S set by the method 0 Comparing, and judging that Sn is more than or equal to S 0 ；
F2: calculating a peak purity value Pn according to the real-time acquired spectrogram and a peak purity threshold value P set by the method 0 Comparing, and judging that the condition Pn is more than or equal to P 0 ；
F3: obtaining a peak height value (absorbance value) Hn from a real-time acquired chromatogram, and a peak height threshold value H set by the method 0 Comparing, and judging that the Hn is more than or equal to H 0 。
3. The preparative liquid chromatography fraction collection method according to claim 2, wherein if any of the determination conditions is not satisfied, the fraction collector remains connected to the waste liquid flow path and does not perform collection.
4. The preparative liquid chromatography fraction collection method of claim 1 wherein in step S7, the retention time t is n If the start-stop time range is not met, the determination condition is not needed, and the fraction collector is kept communicated with the waste liquid flow path and does not collect the waste liquid.
5. A preparative liquid chromatography fraction collection apparatus suitable for use in a preparative liquid chromatography fraction collection method according to any one of claims 1 to 4; the preparative liquid chromatography fraction collection device is characterized by comprising a first solvent bottle, a second solvent bottle, a sample injection pump, a mixer, a preparative chromatographic column, a diode array detector, a fraction collector and a data workstation.
6. A preparative liquid chromatography fraction collector according to claim 5, wherein the first solvent bottle and the second solvent bottle are connected to a mixing tee through infusion lines, respectively, the mixing tee is connected to the mixer, and the infusion lines are provided with a first infusion pump and a second infusion pump, respectively.
7. The preparative liquid chromatography fraction collection device of claim 5 wherein the sample vial is connected to a sample tee via an infusion line; the infusion pipeline is provided with the sample injection pump; the sample feeding tee joint is positioned at the downstream of the mixer; the sample injection tee is also connected with the manual sample injection valve.
8. A preparative liquid chromatography fraction collection device according to claim 5, wherein the outlet of the manual sample injection valve is connected to the inlet of the preparative chromatography column via a liquid delivery line, and the outlet of the preparative chromatography column is connected to the inlet of the diode array detector.
9. The preparative liquid chromatography fraction collector of claim 5 wherein the outlet of the diode array detector is connected to the fraction collection inlet; and outlets of all channels of the fraction collector are respectively connected with corresponding collecting bottles.
10. A preparative liquid chromatography fraction collector according to claim 5 wherein the data workstation is electrically connected to the sample pump, the first infusion pump, the second infusion pump, the diode array detector and the fraction collector, respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211032628.0A CN115531921B (en) | 2022-08-26 | 2022-08-26 | Device and method for collecting preparative liquid chromatographic fraction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211032628.0A CN115531921B (en) | 2022-08-26 | 2022-08-26 | Device and method for collecting preparative liquid chromatographic fraction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115531921A true CN115531921A (en) | 2022-12-30 |
| CN115531921B CN115531921B (en) | 2024-07-16 |
Family
ID=84726261
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211032628.0A Active CN115531921B (en) | 2022-08-26 | 2022-08-26 | Device and method for collecting preparative liquid chromatographic fraction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115531921B (en) |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6002986A (en) * | 1990-11-16 | 1999-12-14 | Shimadzu Corporation | Fraction purity measuring apparatus for chromatogram peak |
| CN1372140A (en) * | 2001-02-27 | 2002-10-02 | 中国科学院大连化学物理研究所 | Fully closed diode array detector and control method thereof |
| JP2004101198A (en) * | 2002-09-04 | 2004-04-02 | Shimadzu Corp | Preparative deice and preparative liquid chromatograph |
| WO2006000840A1 (en) * | 2004-06-28 | 2006-01-05 | Comgenex Rt. | Chromatography purification system |
| US20070278395A1 (en) * | 2004-02-13 | 2007-12-06 | Gorenstein Marc V | Apparatus and Method For Identifying Peaks In Liquid Chromatography/Mass Spectrometry And For Forming Spectra And Chromatograms |
| US20090281282A1 (en) * | 2004-10-19 | 2009-11-12 | Nhs Blood And Transplant | Method for the isolation of haptoglobin |
| CN102124329A (en) * | 2008-08-19 | 2011-07-13 | 株式会社岛津制作所 | Split liquid chromatographic apparatus |
| CN103278590A (en) * | 2013-05-07 | 2013-09-04 | 大连依利特分析仪器有限公司 | Integrated automatic sampling fraction collector |
| CN103487531A (en) * | 2013-01-28 | 2014-01-01 | 中国药科大学 | Efficient preparation method for compound library applicable to natural product high-throughput screening |
| CN104730156A (en) * | 2013-12-23 | 2015-06-24 | 天津市科密欧化学试剂有限公司 | Method for detecting benzenes in chromatographic-grade carbon disulfide |
| CN105259262A (en) * | 2015-10-09 | 2016-01-20 | 中国药科大学 | Novel method for fast finding low-content active ingredients in natural products |
| US20160018370A1 (en) * | 2013-03-04 | 2016-01-21 | Shimadzu Corporation | Preparative separation chromatograph system |
| CN107106930A (en) * | 2014-11-17 | 2017-08-29 | 通用电气健康护理生物科学股份公司 | Method and apparatus for performing liquid chromatography purification |
| CN109932466A (en) * | 2017-12-15 | 2019-06-25 | 中国科学院大连化学物理研究所 | When m- threshold value-peak slope complex controll automatic fraction collector and application |
| CN111551646A (en) * | 2020-05-15 | 2020-08-18 | 云南中烟工业有限责任公司 | Chromatographic peak purity determination method based on mass spectrum similarity |
-
2022
- 2022-08-26 CN CN202211032628.0A patent/CN115531921B/en active Active
Patent Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6002986A (en) * | 1990-11-16 | 1999-12-14 | Shimadzu Corporation | Fraction purity measuring apparatus for chromatogram peak |
| CN1372140A (en) * | 2001-02-27 | 2002-10-02 | 中国科学院大连化学物理研究所 | Fully closed diode array detector and control method thereof |
| JP2004101198A (en) * | 2002-09-04 | 2004-04-02 | Shimadzu Corp | Preparative deice and preparative liquid chromatograph |
| US20070278395A1 (en) * | 2004-02-13 | 2007-12-06 | Gorenstein Marc V | Apparatus and Method For Identifying Peaks In Liquid Chromatography/Mass Spectrometry And For Forming Spectra And Chromatograms |
| WO2006000840A1 (en) * | 2004-06-28 | 2006-01-05 | Comgenex Rt. | Chromatography purification system |
| US20090281282A1 (en) * | 2004-10-19 | 2009-11-12 | Nhs Blood And Transplant | Method for the isolation of haptoglobin |
| CN102124329A (en) * | 2008-08-19 | 2011-07-13 | 株式会社岛津制作所 | Split liquid chromatographic apparatus |
| US20110184658A1 (en) * | 2008-08-19 | 2011-07-28 | Shimadzu Corporation | Preparative Liquid Chromatograph System |
| CN103487531A (en) * | 2013-01-28 | 2014-01-01 | 中国药科大学 | Efficient preparation method for compound library applicable to natural product high-throughput screening |
| US20160018370A1 (en) * | 2013-03-04 | 2016-01-21 | Shimadzu Corporation | Preparative separation chromatograph system |
| CN110064229A (en) * | 2013-03-04 | 2019-07-30 | 株式会社岛津制作所 | Prepare chromatogram arrangement |
| CN103278590A (en) * | 2013-05-07 | 2013-09-04 | 大连依利特分析仪器有限公司 | Integrated automatic sampling fraction collector |
| CN104730156A (en) * | 2013-12-23 | 2015-06-24 | 天津市科密欧化学试剂有限公司 | Method for detecting benzenes in chromatographic-grade carbon disulfide |
| CN107106930A (en) * | 2014-11-17 | 2017-08-29 | 通用电气健康护理生物科学股份公司 | Method and apparatus for performing liquid chromatography purification |
| US20180120271A1 (en) * | 2014-11-17 | 2018-05-03 | Ge Healthcare Bio-Sciences Ab | A Method and Apparatus for Performing Liquid Chromatography Purification |
| CN105259262A (en) * | 2015-10-09 | 2016-01-20 | 中国药科大学 | Novel method for fast finding low-content active ingredients in natural products |
| CN109932466A (en) * | 2017-12-15 | 2019-06-25 | 中国科学院大连化学物理研究所 | When m- threshold value-peak slope complex controll automatic fraction collector and application |
| CN111551646A (en) * | 2020-05-15 | 2020-08-18 | 云南中烟工业有限责任公司 | Chromatographic peak purity determination method based on mass spectrum similarity |
Non-Patent Citations (3)
| Title |
|---|
| ZENG, L: ""Automated analytical/preparative high-performance liquid chromatography mass spectrometry system for the rapid characterization and purification of compound libraries"", 《JOURNAL OF CHROMATOGRAPHY A》, 23 January 1998 (1998-01-23) * |
| 周杰: ""鹿茸蛋白HPLC指纹图谱研究及在线二维液相色谱的建立和应用"", 《中国优秀硕士学位论文全文数据库》, 15 November 2011 (2011-11-15) * |
| 李彤: ""高效液相色谱和超高效液相色谱仪器的一些最新发展"", 《色谱》, 8 October 2015 (2015-10-08) * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115531921B (en) | 2024-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102612648B (en) | Multi-function selector valve, fully automatic multi-function liquid chromatography device having same, and method for sample analysis using same | |
| CN107073358B (en) | For two to RPLC-SFC chromatography system and method | |
| Schomburg et al. | Aspects of double-column gas chromatography with glass capillaries involving intermediate trapping | |
| US10753915B2 (en) | Methods for analysis of phase-I and phase-II metabolites and parent compounds without hydrolysis | |
| US20200406165A1 (en) | Multi-dimensional chromatographic system for analyzing multiple sample components | |
| CN103940941A (en) | On-line analysis system for automatic pretreatment by adopting ultra high performance liquid chromatography/mass spectrum | |
| CN103235074B (en) | High performance liquid chromatography online analysis method and application thereof | |
| CN103308621A (en) | Method for detecting 25(hydroxyl)vitamin D by using high-pass liquid chromatography-tandem mass spectrometry | |
| US11408865B2 (en) | Sample injector | |
| CN110208401A (en) | Solid phase is dehydrated extraction-supercritical fluid chromatography-mass spectrum on-line analysis system and method | |
| WO2021249347A1 (en) | Static sample injection and fraction collection apparatus for use with liquid chromatograph | |
| US3860393A (en) | Automated system for identifying traces of organic chemical compounds in aqueous solutions | |
| CN110064229A (en) | Prepare chromatogram arrangement | |
| CN104678014B (en) | A kind of Two-dimensional Liquid Chromatography | |
| CN115531921A (en) | Preparative liquid chromatography fraction collecting device and method | |
| Válcarcel et al. | Coupling continuous sample treatment systems to capillary electophoresis | |
| CN110146611B (en) | Method for rapidly identifying chemical components in donkey-hide gelatin blood-enriching particles | |
| CN208654099U (en) | Transformer oil gas analysis Special instrument | |
| JPWO2016098169A1 (en) | Liquid chromatograph | |
| CN217981369U (en) | Two-dimensional liquid chromatography separation system | |
| CN114460207B (en) | Identification method of radix achyranthis bidentatae adulterated medicinal material and medicinal slices of radix achyranthis bidentatae | |
| EP0359322A2 (en) | Liquid chromatography | |
| CN215653921U (en) | Tobacco essence extraction and separation device | |
| CN112180021B (en) | Liquid Chromatography System | |
| CN206489129U (en) | Integral type form and macroanalysis atomic fluorescence sample introduction and pretreatment system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |